T cell independent mechanism for copolymer-1-induced neuroprotection

T cell independent mechanism for copolymer-1-inducedneuroprotection

Jianuo Liu1,2,7, Thomas V. Johnson1,2,3, Jamie Lin1,2,7,Servio H. Ramirez2,6,7, Tatiana K. Bronich4, Steve Caplan5,Yuri Persidsky2,6,7, Howard E. Gendelman2,7 and Jonathan Kipnis1,2,3,7,8

1 Laboratory of Neuro-Immune Regulation, University of Nebraska Medical Center,Omaha, Nebraska, USA

2 Departments of Pharmacology and Experimental Neuroscience, University ofNebraska Medical Center, Omaha, Nebraska, USA

3 Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha,Nebraska, USA

4 Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska,USA

5 Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha,Nebraska, USA

6 Pathology and Microbiology, University of Nebraska Medical Center, Omaha,Nebraska, USA

7 Center for Neurovirology and Neurodegenerative Disorders, University of NebraskaMedical Center, Omaha, Nebraska, USA

8 Department of Neuroscience, University of Virginia, Charlottesville, Virginia, USA

Despite active investigation of copolymer-1 (Cop-1) for nearly 40 years the mechanismsunderlying its neuroprotective properties remain contentious. Nonetheless, currentdogma for Cop-1 neuroprotective activities in autoimmune and neurodegenerativediseases include bystander suppression of autoimmune T cells and attenuation ofmicroglial responses. In this report, we demonstrate that Cop-1 interacts directly withprimary human neurons and decreases neuronal cell death induced by staurosporine oroxidative stress. This neuroprotection is mediated through protein kinase Ca and brain-derived neurotrophic factor. Dendritic cells (DC) uptake Cop-1, deliver it to the injurysite, and release it in an active form. Interactions between Cop-1 and DC enhance DCblood brain barrier migration. In a rat model with optic nerve crush injury, Cop-1-primed DC induce Tcell independent neuroprotection. These findings may facilitate thedevelopment of neuroprotective approaches using DC-mediated Cop-1 delivery todiseased nervous tissue.

Introduction

Copolymer-1 (Cop-1) was developed to mimic theencephalitogenic epitope of myelin basic protein(MBP). However, when administered in completeFreund's adjuvant it failed to induce experimentalautoimmune encephalitis (EAE). Surprisingly, Cop-1suppressed EAE when administered with MBP [1–4].These observations led to the development of Cop-1 as atreatment for relapsing-remitting multiple sclerosis

Correspondence: Dr. Jonathan Kipnis, Department of Neu-roscience, University of Virginia, Charlottesville, VA 22908, USAFax: +1-434-982-4380e-mail: [email protected]

Received 17/4/07Revised 7/8/07

Accepted 14/9/07

[DOI 10.1002/eji.200737398]

Key words:Dendritic cells� Inflammation

� Neuroimmunology� T cells

Abbreviations: BBB: blood brain barrier � BDNF: brain-derivedneurotrophic factor � Cop-1: copolymer-1 � POMA: blockcopolymer poly-ethylene oxide/poly-methacrylic acid �PKCa: protein kinase Ca � RGC: retinal ganglion cell �tBu-OOH: tert-butyl hydroperoxide solution

Eur. J. Immunol. 2007. 37: 3143–3154 Immunomodulation 3143

f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

[5–8]. Over the past three decades, scientists have triedto determine how Cop-1 exerts its therapeutic effects [2,4, 9, 10]. Cop-1-specific T cells partially cross-react withMBP, leading to the secretion of neurotrophic and anti-inflammatory factors such as brain derived neurotrophicfactor (BDNF), glial cell-derived neurotrophic factor,TGF-b, and IL-10 [4, 11–13]. Cop-1 elicits neuroprotec-tive responses in animal models of neuroimmune,neurodegenerative, metabolic, and traumatic disorders[14]. This includes, but is not limited to, optic nerveinjury [11], head trauma [15], Parkinson's disease [12],Alzheimer's disease [16], HIV-1-encephalitis [13],glaucoma [17], and experimentally induced encephalitis[18].

The mechanism(s) underlying Cop-1-induced neu-roprotection were attributed to T cells. T cells modulatemicroglial and astrocyte responses and as such attenuatetoxic inflammatory activities and neurodegeneration[11, 12, 19]. Indeed, the mechanism underlying T cell-induced neuroprotection is linked to modulation ofmicroglial function [20] and increased production ofneurotrophins at the site of injury [11, 12]. After centralnervous system (CNS) injury microglia commonlydisplay a neurotoxic phenotype with local productionof glutamate and increased expression of nitric oxidesynthase (iNOS), pro-inflammatory cytokines, quinoli-nic acid and arachidonic acid and its metabolites [21,22]. T cells can induce a neuroprotective microglialphenotype [20].

Under certain conditions with progressive neurode-generation the Cop-1 neuroprotective response is seenbefore an adaptive immune response is generated [15,17, 23], suggesting that Cop-1 induced neuroprotectionmay be, at least in part, T cell independent. We nowdemonstrate that Cop-1 treatment of primary humanneurons intoxicated with staurosporine or reactive-oxygen species significantly decreases cell death. Thisneuroprotection is associated with increased BDNF andis inhibited by an inhibitor of protein kinase C alpha(PKCa) signaling pathway. Uptake of Cop-1 by DCincreases migration of DC across the blood-brain barrier.The slow release of Cop-1 in its active form from DCsuggests Cop-1 can be delivered by DC to injured brainareas and be released there. Using rat model with opticnerve crush injury we demonstrate here that Cop-1-primed DC induce neuroprotection without eliciting aT-cell response. Moreover, Cop-1 treatment was neuro-protective following optic nerve crush injury in athymicnude rats, further supporting a T cell-independentmechanism of Cop-1 action.

Results

Direct Cop-1-induced neuroprotection

To determine if Cop-1 directly induces neuroprotection,primary human neurons were intoxicated with staur-osporine and immediately treated with escalating Cop-1doses. Cop-1 induced a concentration-dependent neu-roprotection as determined by significant reduction inTUNEL positive apoptotic cells 24 h after treatment(Fig. 1a). Block copolymer poly-ethylene oxide/poly-methacrylic acid, (POMA) used as a control did notdemonstrate any effects on neural survival. Represen-tativemicrographs of TUNEL positive cells are illustratedin Fig. 1b. In addition, anti-dsDNA ELISA tests showedan inverse relationship between Cop-1 concentrationand dsDNA levels (Fig. 1c).

Staurosporine down-regulates the activation ofPKCa, a major neuronal survival factor, and otherkinases, leading to neuronal apoptosis [24, 25]. Toexamine whether Cop-1 affects neuroprotection throughPKCa we used tert-butyl hydroperoxide solution (tBu-OOH) to induce oxidative-stress mediated neuronaldeath. Cop-1 significantly protected neurons from tBu-OOH-induced neurotoxicity. This effect was abrogatedby the PKCa-specific inhibitor Go6976 (Fig. 1d), sug-gesting that Cop-1 neuroprotective activities weremediated through PKCa.

Cultures of primary human neurons commonlycontain *10% astrocytes. To exclude an astrocyte rolein the observed effects of Cop-1 on neuronal survival weused theMES 23.5 dopaminergic neuronal cell line. MES23.5 cells treated with 50 lM staurosporine with orwithout Cop-1 demonstrated that staurosporine neuro-toxicity was reduced by Cop-1 in a dose-dependentmanner (Fig. 1e), providing further support for directCop-1 neuroprotection.

BDNF, a survival factor downstream from PKCa[26–28], mediates neuronal survival during develop-ment and after neural injury [29–32]. To verify directinteraction between Cop-1 and neurons, Cop-1 wasincubated for 24 h with primary human neurons andneurons were then immunolabeled for BDNF expres-sion. Quantitative immunofluorescence assays showedabout twofold increase in neuronal expression of BDNFfollowing Cop-1 treatment as compared to POMA or notreatment (Fig. 1f). Representative confocal fields arepresented in Fig. 1g. These results were furtherconfirmed by ELISA tests of neuronal culture fluids(Fig. 1h).

Jianuo Liu et al. Eur. J. Immunol. 2007. 37: 3143–31543144


Cop-1 affects DC secretory and migratoryfunctions

We hypothesized that Cop-1 may be taken up at itsinjection site by DC then delivered across the blood brainbarrier (BBB) to the CNS where it would directly affect

neurons. We examined Cop-1 uptake and release by DCover time. Human monocytes were differentiated intoDC in the presence of granulocyte-macrophage colonystimulating factor (GM-CSF) and IL-4. Differentiated DCwere incubated with FITC-labeled Cop-1 for 24 h andthen immunolabeled with CD11c (membrane marker of

Figure 1. Cop-1 directly induces neuroprotective effect on primary neuronal cultures. Human neurons were treated with orwithout Cop-1 in presence of staurosporine for 24 h. TUNEL staining and ELISAwere performed to analyze neuronal apoptosis. (a)Quantitative analysis of the TUNEL staining revealed significant reduction in apoptotic cells in Cop-1-treated neuronal culturerelative to control neurons. (b) Representative fields showing apoptotic cells (scale bar equals 50 lm). (c) ELISAanalysis of a double-stranded DNA fragments in primary human neuron cultures treatedwith increased concentration of Cop-1. (d) TUNEL staining oftBu-OOH-intoxicated neurons treatedwith Cop-1 in the presence of PKCa inhibitor, Go6976. (e) Quantitative analysis of the TUNELstaining of dopaminergic neuronal cell line showed reduced apoptotic cell numbers in a dose response to Cop-1. (f, g)Immunolabeling for BDNF in Cop-1-treated and control human neurons. Quantification of fluorescencewas performed using NIHimage pro software. Representative micrographs are shown (scale bar equals 50 lm). (h) Bar graph represents quantitative ELISAresults of conditioned media of staurosporine-intoxicated neurons with or without Cop-1 treatment. ANOVA with post hocBonferroni test for pair comparison was used for statistical analysis (in (h) Student's t-test was used). Asterisks indicatesignificance (*p <0.05; **p <0.01; ***p <0.001).



myeloid lineage). FITC-labeled Cop-1 was not localizedwith membrane or nuclear (DAPI) staining, but ratherwas found in the cytoplasm. Representative confocalimages are shown in Fig. 2a. To determine a time pointfor which Cop-1 is retained in DC, we incubated DCwithFITC-labeled Cop-1, washed after 24 h and fresh mediawere added. Cell samples were analyzed by FACS every24 h. Some FITC labeling remained in cells up to 72 hafter incubation (Fig. 2b). Conditioned media of thewashed DC was collected every 24 h and replaced withnew Cop-1-free medium. Concentrations of FITC-labeled peptides in the conditioned media weremeasured. The largest concentration of FITC-labelingwas found in the medium collected 24 h after incuba-tion. Additional release of FITC-positive peptides intothe medium was detectable over the next 48 h (Fig. 2c).

The size of the FITC-labeled molecules found in theconditioned media is not yet known. Future studies willaim to identify the minimal active unit of Cop-1 asreleased from DC. However, to assess whether the

released FITC-labeled molecules possess any biologicalactivity (neuroprotective), we incubated staurosporine-intoxicated neurons with supernatants obtained fromthe cultured Cop-1-primed DC. Supernatants collectedafter the first 24 h of cell cultivation showed neuropro-tective activities (Fig. 2d). This observation correlatedwith the concentration of FITC labeling detected in theconditioned media. Conditioned media obtained fromPOMA-incubated DC after 24 h did not affect neuronalsurvival.

To determine if Cop-1 affects DC morphology welabeled the cells with phalloidin to examine cytoskeletalchanges [33]. Untreated circular-shaped DC changed toamoeboid cells when treated with Cop-1 (Fig. 3a). Incontrast, proliferative responses induced by Cop-1treated DC cultured with autologous CD3+ T cells werenot altered, suggesting that Cop-1 did not affectcostimulatory properties of DC (Fig. 3b). As Cop-1may induce expression of growth factors by DC and,thus, indirectly provide neuroprotection, we performed

Figure 2. Cop-1 released from DC confers neuro-protection on primary neurons. (a) DCwere exposedto FITC-labeled Cop-1 for 24 h and immunolabeledwith anti-CD11c mAb and analyzed by confocalmicroscope (scale bar equals 10 lm). (b) DC wereloadedwith FITC-labeled Cop-1 and harvested every24 h for FACS analysis. The results are representa-tive of four independent experiments. (c) DC wereincubatedwith FITC-labeled Cop-1 for 24 h, washed,and media was collected every 24 h and replacedwith new Cop-1 free media. Concentrations of FITClabeling in DC cultures was measured at 24, 48 and72 h. (d) The media collected in (c) were examinedfor neuroprotection by TUNEL labeling on stauros-porine-intoxicated neurons. POMA was used as anegative control copolymer in these experimentsand did not have any effect on neuronal survival.The effect was significant in the supernatant fromthe first 24 h of incubation. ANOVA with post hocBonferroni test for pair comparison was utilized forstatistical analysis. Asterisks indicate significance(*p <0.05; **p <0.01).



protein array analysis of replicate conditioned mediaobtained from Cop-1-primed and control DC. Nodifferences in any of the available neurotrophins orgrowth factors were detected in Cop-1 treated DC (datanot shown).

We next examined whether Cop-1 affects DCmigration across a BBB laboratory model [34, 35]. DClabeled with the fluorescence marker Calcein-AM, wereplaced in the upper chamber of BBB constructscontaining primary human brain microvascular en-

dothelial monolayers. After 4 h, the relative fluores-cence from the DC that migrated to lower chamber wasmeasured. Fig. 3c shows that priming DC with Cop-1enhanced their transendothelial migration, whereasPOMA did not have any effect. To examine a possibleeffect of released Cop-1 on endothelial cells and thus apotential indirect effect of Cop-1 on DC migration, wetreated endothelial cells with 30 lg/mL of Cop-1 for 4 h,and thenwashed, applied new (Cop-1 free) medium andexamined migration of DC. No difference in DC

Figure 3.Cop-1 induces changes in DC. (a) Immunolabeling of DCwith rhodamine phalloidin indicated a Cop-1-induced change inDC cell phenotype (shape). Representative confocal images are shown. (b) DC pre-treated with Cop-1 for 24 h, were cultured withCD3+ T cells in the presence of 1 lg/mL of anti-CD3 mAb. T cell proliferation was measured via [3H]thymidine incorporation intriplicate assays. (c, d) Results from quantitative migration assays using an in vitro BBB model that utilizes primary human brainmicrovascular endothelial cells (BMVEC) and DC labeled with Calcein-AM fluorescent marker are shown. (c) The relativefluorescence from control and DC (2 � 105/mL) primed with either Cop-1 or POMA was measured after 4 h of migration (asdescribed in Materials and methods). (d) Endothelial monolayers were treated with or without Cop-1 for 4 h, and then washed,followed by the addition of fluorescently labeled DC. Transendothelial migration of DC was examined as in (c). The data arerepresented as the number of migrated DC, which was determined from the relative fluorescence of known numbers of labeledDC. (e) The GTPase activity of RhoA in lysates from DC pulsed with Cop-1 or unpulsed was analyzed. b-actin blotting wasperformed on the same membranes to address the total amount of loaded protein in each group. (f) Following unilateral opticnerve crush injury rats were subcutaneously injected with 10 � 106 111Indium-labeled DC (treated with Cop-1 or control). One daylater, optic nerves were excised and measured for 111Indium oxine by c-scintillation spectrometry. (g) Servical lymph nodes weresliced and autoradiographed. In Cop-1-loaded DC-treated rats significant radiolabeling is evident in the lymph nodes as opposedto control DC treated animals. ANOVA with post hoc Bonferroni test for pair comparison was utilized for statistical analysis.Asterisks indicate significance (**p <0.01).



migration was determined when endothelial cells weretreated with Cop-1 (Fig. 3d), suggesting that anenhanced migration of DC after Cop-1 treatment isdirectly an effect of Cop-1 on DC.

Migration of DC is generally associated with activa-tion/phosphorylation of small GTPases, with a conse-quent change in cell phenotype [36, 37]. Analysis of theeffect of Cop-1 treatment on RhoA disclosed that theGTPase activity of RhoA in lysates from Cop-1-primedDC was significantly higher than in untreated controls

(Fig. 3e), suggesting that the observed changes in cellshape and migration might be related to GTPase activity.

To address the ability of Cop-1 to increase migrationof DC in vivo, we labeled DC with 111Indium and injectedthem into rats with optic nerve injury. Twenty-fourhours after injection, optic nerves were excised andradioactivity measured. Significantly higher cpm weredetected in injured nerves than in non-injured, and Cop-1-treated DC led to a further increase in radioactivity(Fig. 3f), suggesting that Cop-1-treated DC are better

Figure 4. DC loaded with Cop-1 attenuate degeneration of injured optic nerve fibers in vivo. (a) Rats with optic nerve crush injurywere subcutaneously injected with bone marrow derived control DC or DC primedwith either Cop-1 or POMA (30 lg/mL), or withPBS. Two weeks later the RGC were retrogradely labeled with a fluorescent dye to the optic nerve distal to the site of injury. Afteradditional 5 days, whole retinas were excised and flat-mounted. Labeled RGC were counted under fluorescent microscope. (b)Representative micrographs of flat-mounted retinas are shown (from DC-Cop and DC-POMA treated animals) as observed underfluorescent microscope. (c) Single-lymphocyte suspension was prepared from lymph nodes of DC (control of Cop-1 activated)treated rats (subcutaneous injection) and stimulated with soluble Cop-1 (15 lg/mL), OVA (25 lg/mL) or ConA (5 lg/mL) in vitro.Lymphocyte proliferationwasmeasured by [3H]thymidine incorporation in triplicate assays and cpm counts of each treatment foreach group are presented. (d) A single sub-cutaneous injection of Cop-1 intowild-type or athymic nude rats immediately after theinjury induces significant neuroprotection. Bar graphs represent RGC counts/mm2. (e) Injury was inflicted in wild type rat andanimals were treated as in (a). Optic nerves were excised 7 days after the injury and examined for BDNF immunoreactivity.Quantification of fluorescence intensity per examined area was performed using NIH Image Pro software. At least six slices wereexamined from each animal from at least four animals per group. (f) Representative micrographs of optic nerves immunolabeledfor BDNF are shown from rats treated with DC primed with either Cop-1 or POMA at 30 lg/mL concentration (scale bar equals100 lm). (g) Western blots for BDNF and b-actin from injured and contralateral retinas 7 days following optic nerve crush injuryand a subsequent treatment with Cop-1 or POMA primed DC. ANOVA with post hoc Bonferroni test for pair comparison wasutilized for statistical analyses. Asterisks indicate significance (*p <0.05; **p <0.01; ***p <0.001).



migratory cells in vivo. It is possible, however, that theobtained radioactivity is not a result of DC migration tothe site of injury but rather a passive transmigration ofCop-1 containing vesicles released from DC. Autoradio-graphy of cervical lymph nodes revealed significantlyincreased radioactivity in rats injected with Cop-1loaded DC as compared to control DC (Fig. 3g), furthersupporting the ability of Cop-1 to increase DCmigration.

Cop-1-DC attenuate neurodegeneration afteroptic nerve crush injury

Using a rat model of acute optic nerve crush [38], weinvestigated the neuroprotective effects of Cop-1delivered via bone marrow-derived DC to CNS injuredsites. The right optic nerves of adult male rats werecrushed, and the rats were immediately injectedsubcutaneously with DC that had been primed withCop-1 or POMA for 24 h after their differentiation (n=8in each group) or with unprimed control DC (n = 8).Rats injected with PBS immediately following the crushinjury were included as an additional control group (n=8). Two weeks after the injury, retinal ganglion cellswere retrogradely labeled with a fluorescent dye applieddistal to the site of the injury. After additional 5 days therats were euthanized, and their retinas excised and flat-mounted for microscopic examination. The retinalganglion cell (RGC) survival was assessed by anexaminer blinded to group identity. Injection of controlPOMA primed DC did not affect the outcome relative tothe PBS-treated group, whereas the Cop-1-primed DCprovided significant neuroprotection compared to allcontrol groups (Fig. 4a). Representative micrographsfrom the retinas are shown in Fig. 4b.

A single subcutaneous DC vaccination does notinduce a strong CD4+ T cell response [39–42]. To beconfident that the observed beneficial effect after DCvaccination was not due to a neuroprotective Cop-1-specific T-cell response induced by DC vaccination [11],we inoculated rats with PBS, with unprimed DC, or withCop-1-primed DC. One week later we examined theharvested lymphocyte response to Cop-1 and otherproteins in vitro. Rats treated with Cop-1-pulsed DC didnot show a higher T-cell response than the other twogroups (Fig. 4c), indicating that the beneficial effect ofthe DC vaccination was not attributable to a Cop-1-specific T-cell reaction.

To further examine whether Cop-1-induced neuro-protection was independent of T cell activities, opticnerve-injured athymic nude rats were treated with asingle dose of Cop-1 (n = 8) immediately followinginjury. A control group was treated with PBS (n = 8).Cop-1-treated immunodeficient mice showed significantneuroprotection (Fig. 4d). Wild-type rats treated simi-larly showed significant neuroprotection with Cop-1

treatment. As expected from our previous studies, Tcell-deficient rats showed lower neuron survival due to lackof endogenous neuroprotective T cell mechanism;therefore, the Cop-1-treated group did not reach thevalue of wild-type treated rats. However the magnitudeof Cop-1 effect in both strains was similar (140 � 26%for wild types vs. 135� 18% for athymic nudes; n= 6–8rats in each group; Student's t-test was used forstatistical analysis).

To address a possible in vivo mechanism underlyingDC-Cop-1-induced neuroprotection we examined ex-pression of BDNF in optic nerves of treated and controlrats. Animals were injured and treated with eitherPOMA or Cop-1-primed DC, control DC or PBS. Sevendays after treatment optic nerves were excised andexamined for BDNF expression. BDNF immunolabelingof injured optic nerves showed a significant increase inBDNF protein levels in DC-Cop-1-treated mice ascompared to control groups (Fig. 4e). Representativemicrographs of POMA and Cop-1 primed DC-treatedmice are shown (Fig. 4f). Retinas of rats treated witheither POMA or Cop-1-primed DC following optic nervecrush injury were examined for BDNF expression.Significantly higher expression of BDNF was evidentin DC-Cop-1-treated rats in both injured and contral-ateral retina as was revealed by Western blot examina-tion (Fig. 4g).

Discussion

We now demonstrate that Cop-1 can elicit directneuroprotective activities associated with increasedlevels of neuronal BDNF. Uptake of Cop-1 by DCimproved migration across the BBB and was associatedwith RhoA activation. Injection of Cop-1-pulsed DC afteroptic nerve injury significantly increased neuronalsurvival without inducing a Cop-1-specific T-cellresponse. Athymic nude rats with optic nerve crushinjury also benefited from Cop-1 treatment, supportinga Tcell-independent Cop-1 mechanism for neuroprotec-tion.

Bystander suppression of Cop-1-reactive T cells anddirect Cop-1-induced displacement of myelin peptidesfrom the class-II MHC groove of antigen-presenting cellshave both been proposed as underlying mechanisms forCop-1-mediated anti-inflammatory activities [43, 44].Cop-1 also protects neurons under acute and chronicneurodegenerative conditions [14].

Although the earlier studies showed that autoim-mune T cells could protect injured neurons fromongoing degeneration, this beneficial effect was coun-terbalanced by their propensity for inducing destructiveautoimmunity [45]. To overcome this deleterious effect,Tcells that cross-react weakly with autoantigens without



inducing an autoimmune disease were tested forneuroprotective activities. Cop-1 is capable of activatingT cells that cross-react with autoantigens, therebyconferring neuroprotection without inducing harmfulautoimmune response [11]. While Cop-1-inducedneuroprotection could be obtained by passive T celltransfer, later studies demonstrated that more signifi-cant neuroprotection is achieved by subcutaneousinjection of naked Cop-1 (without adjuvant) [17]. TheT cell-mediated immune response elicited by this lattertype of immunization is weak [46–48]. These resultsquestioned the role of T cells in Cop-1-mediatedneuroprotection, and led us to postulate additionalmechanisms mediating Cop-1-induced neuroprotection.Based on the observations made in this report andpreviously published works [11, 12, 16, 18, 49], wesuggest that in neurodegenerative conditions, Cop-1 canaffect neuroprotective outcomes by at least twoindependent mechanisms: (i) DC, directly deliveringCop-1 to injury sites where Cop-1 is released and directlyaffecting neurons, and (ii) induction of T cell-mediatedresponse. This dual effect likely produces the rapid andlong-lasting effect of Cop-1 during disease [11, 12,15–17, 23, 50–52].

Several studies have demonstrated a significanteffect of Cop-1 on the innate cells of the immunesystem, such as macrophages, DC, and monocytes[53–56]. Recent studies have characterized the pheno-types of these cells from Cop-1-treated patients [54, 57].Monocytes and DC from these patients showed analtered cytokine profile and altered expression of HLA-DR and costimulatory molecules [54]. The results of thepresent study further demonstrate the effect of Cop-1 oninnate immune cells. We showed a significant increase inthe ability of Cop-1-primed DC to migrate across theBBB, activate RhoA, and change cell shape. Theidentities of specific intracellular signaling moleculesthat might interact with Cop-1 have not been deter-mined. Although our results strongly support a notion ofimproved trans-endothelial migration of DC followingpriming with Cop-1, the obtained results in vivomay alsosuggest that rather than DC migrated to the site ofinjury, vesicles released from DC and containing Cop-1,passively migrated through the BBB and thus inducedthe neuroprotective effect. To address these twopossibilities in vivo, future studies will aim on trackinglabeled DC using imaging techniques and/or co-registration of radiographywith immunohistochemistry.

Conditioned medium from Cop-1-primed DC had asignificant neuroprotective effect on primary humanneuronal cultures exposed to staurosporine toxicity. Todetermine whether Cop-1 treatment induces macro-phages and DC to release factors that mediateneuroprotection, we examined the growth factorcontent of the conditioned medium. Protein-array

analysis showed no significant differences in neurotro-phins or other growth factors in Cop-1-treated cellscompared to untreated controls. Interestingly, we foundthat FITC-labeled molecules were released from DC andaccumulated in culture fluids. The concentration of FITClabeling correlated with the neuroprotective propertiesof the conditioned media. We assume that FITC-labeledmolecules are active breakdown products of Cop-1.Alternatively, naked FITC molecules could be releasedfrom DC proportionally to cleaved Cop-1 molecules,which explains the above-mentioned correlation be-tween FITC labeling and neuroprotection of theconditioned media. Future studies will aim to identifythe shortest active unit of Cop-1 and to characterize Cop-1 breakdown products in the DC conditioned media.

Cop-1, when applied to neuronal cultures concur-rently with a neurotoxin such as staurosporine or tBu-OOH, protected neurons from degeneration. We ob-served dependence of the neuroprotective effect of Cop-1 on PKCa following oxidative stress. Activation of PKCatriggers a survival mechanism and underlies theneuroprotective mechanisms of several drugs. As anexample, Rasagiline, a drug that was recently approvedfor Parkinson's disease, confers its neuroprotective effectvia PKCa [58]. Activation of PKCa induces BDNFexpression from neural cells [28]. We also found BDNFinduction in Cop-1-treated neurons and in vivo at the siteof injury and in the retina following treatment with Cop-1-pulsed DC.

The large size of the Cop-1 polypeptide prevents itfrom crossing the BBB. Using an artificial BBB, weshowed that DC cells could potentially deliver Cop-1 tothe brain. Alternatively, Cop-1 can be broken down tosmaller polypeptides by DC and released into the bloodstream. These Cop-1-breakdown products may them-selves be neuroprotective, and may be capable ofcrossing the BBB to mediate their neuroprotectiveeffects. Moreover, these DC can amplify a T cell-mediated immune response in the CNS [59], whichcan further potentiate neuroprotective effects of Cop-1.

These data, taken together, provide a novel mechan-ism of Cop-1 mediated neuroprotection, enhancing itsutility for treatment of neurodegenerative and neuroin-flammatory disorders.

Materials and methods

Animals

Inbred female adult Lewis rats (8–12 weeks old) were suppliedby Jackson Laboratories (Bar Harbor, Maine). Athymic nuderats were purchased from the National Institutes of Health(NIH; Bethesda, MD). Animals are housed in a light- andtemperature-controlled room and are matched for age in eachexperiment. Animals were handled according to the regula-



tions formulated by our Institutional Animal Care and UseCommittee at the University of Nebraska Medical Center.

Copolymer-1

Cop-1 (cat no. P1152) was purchased from Sigma Aldrich (St.Louis, MO). Cop-1 was conjugated with FITC (FITC-Cop-1)using standard procedure [60]. Briefly, 0.05 mL of 1% solutionof FITC in DMF was added to 0.9 mL of the Cop-1 solution(4 mg/mL) in 0.1 M sodium carbonate buffer, pH 9.0, andstirred overnight at room temperature. The conjugate waspurified from non-reactive dye by gel filtration on SephadexG-25 column (NAP-10, Amersham Biosciences) following thedialysis against distilled water and lyophilization. Fluores-cence spectral characteristics of FITC-labeled Cop-1 weredetermined at kex = 488 nm and kem = 518 nm usingShimadzu RF5000 spectrofluorimeter.

Purification of T cells and T cell subsets

To obtain purified Human CD3+ or CD4+ T cells, human panT cell isolation kit or human CD4 isolation kit were used basedonmanufacturer's recommendations. Highly pure CD3+ Tcellsor CD4+ T cells were obtained via passing autoMACSTM

Columns (Miltenyi Biotec, Bergisch Gladbach, Germany).

Preparation of monocyte-derived DC

Human monocytes and lymphocytes were isolated as pre-viously described [61]. Briefly, peripheral blood of healthyhuman donors was used as a source and monocytes werepurified by counter-current centrifugal elutriation (AmershamBioscience). DC were generated from isolated monocytes. Inbrief, monocytes were cultured at 2 � 106/mL in RPMI 1640with 10% human serum, 100 U-100 lg/mL penicillin-strepto-mycin, 50 ng/mL of rh GMCSF and 20 ng/mL of rh IL-4 for7 days. Medium was changed every third day. After 7 days inculture, DC were activated for additional 24 h with eithernone, Cop-1, or irrelevant control copolymer, POMA.

Proliferative response in naive T cells and T cell subsets

DC were seeded (3 � 104/well) in 96-well flat-bottom plates(Corning) and cultured with CD3+ T cells in addition to thepresence of 1 lg/mL purified anti-human CD3 mAb (clone:Hit3a, BD PharMingen) in complete culture media (RPMI 1640supplemented with 5% human AB sera, 2 mM L-glutamine,100 U/mL penicillin/100 lg/mL streptomycin, 5 mM HEPES,0.5 mM sodium pyruvate, and 0.05 mM nonessential aminoacids). Cell proliferation was assayed by [3H]thymidine(Amersham Biosciences, Arlington Heights, IL) incorporationduring the last 16 h of a 3-day cell cultivation and radioactivitydetermined in a TopCount NXT liquid scintillation counter(Packard).

Isolation of human fetal neurons

Human fetal neurons were prepared from aborted fetal braintissue (gestational age 90–120 days) in compliance with theUniversity of Nebraska Medical Center and National Institutes

of Health ethical guidelines. Briefly, human fetal brain tissuewas digested with 0.25% trypsin plus DNase. After washing,cells were seeded in poly-D-lysine-coated 48-well plates at adensity of 1 � 105/well and maintained in Neurobasal mediasupplemented with B27. Five days later, 10 lg/mL of5-fluorodeoxyuridine was added to inhibit proliferation ofdividing astrocytes and fibroblasts. The purity of neural cellswas determined by staining with MAP-2 mAb (MatureNeuronal Marker). After 2 weeks more than 80% of MAP-2-positive cells were obtained in neural cultures.

TUNEL assay

TUNEL assays were performed using an 'In Situ Cell DeathDetection' kit, AP according to the manufacturer's instructions.Apoptotic cells identified with TUNEL were normalized by40,60-diamidino-2-phenylindole (DAPI) nuclei staining. TU-NEL-positive cells were scored blindly to determine thepercentage of TUNEL+/DAPI+ cells. We developed a neuronalapoptosis index by dividing the number of counted TUNEL+

cells by total DAPI+ cells.

ELISA dsDNA

DNA fragmentation was detected by Cell Death DetectionELISA (Roche, Indianapolis, IN) following the manufacturer'sinstructions. Briefly, neuronal cultures were lysed and placedinto a streptavidin-coated 96-well plate. A mixture of anti-histone-biotin (mAb, clone H1) and anti-DNA-POD (mAb cloneMCA-33) was added and incubated. POD was determinedphotometrically using ABTS as substrate.

Human BMVEC cell culture

Primary human brain microvascular endothelial cells(BMVEC) were isolated from tissue derived from surgicalremoval of epileptogenic cerebral cortex, as describedpreviously [34] and provided by Drs. Marlys Witte andMichael Bernas (University of Arizona, Tucson, AZ). Theprocedures were approved by the University of NebraskaMedical Center Institutional Review Board. BMVEC weremaintained in DMEM/F-12 media containing 10% horseserum, endothelial growth factor (0.1 mg/mL, R&D Systems,Minneapolis, MN), heparin (100 mg/mL, Sigma, St. Louis,MO), amphotericin B (2.5 lg/mL, Invitrogen, Carlsbad, CA),and gentamicin (50 lg/mL, Invitrogen). Only BMVEC of lowpassage were used. For barrier formation, the cells were platedconfluent in a given culture vessel and maintained in mediacontaining the above components but lacking the rEGF, whichallows BMVEC to display typical morphological and functionalbarrier characteristics after 3 to 5 days in culture.

Quantitative transendothelial migration assay

The transendothelial migration assay was a fluorescence-basedassay employing an in vitromodel of the BBB. Briefly, primaryhuman BMVEC at 2.5 � 104 cells/insert were grown toconfluence on collagen (type I) coated FluoroBlokTM tissueculture inserts (with 3-micron pores, BD, Franklin Lakes, NJ).Manual readings of transendothelial resistance (TEER) were



then taken with a voltmeter (EVOM, World PrecisionInstruments, Sarasota, FL) to ensure barrier formation asdescribed earlier [62]. Thereafter, endothelial cells were leftuntreated or treated with either COP-1 or the control POMA atthe indicated concentrations for 4 h. Alternatively, the DC butnot the endothelial cells were treated with the test compoundsalso for 4 h. Prior to initiation of migration, the DC werelabeled with calcein-AM at 5 lM/1 � 106 cells for 45 min at37�C. After the corresponding treatments, the BMVECmonolayers and the DC were washed of all test compounds.The DC were then added at 2 � 105 cells/insert to the upperchamber of the insert and migrated towards recombinanthuman monocyte chemoattractant protein (MCP)-1 (CCL2/MCP-1, 30 ng/mL, R&D) placed into the lower chamber.Migrations were allowed to continue for up to 4 h at 37�C. Themigrations were monitored by taking measurements of therelative fluorescence using a SpectraMax M5 fluorescenceplate reader (Molecular Devices, Sunnyvale, CA) of the labeleddendritic cells found in the lower chamber. The measurementscollected were then plotted against standard curves fromrelative fluorescence of known numbers of DC. The data arerepresented as the average number of migrating dendritic cells� SEM.

Immunocytochemistry

Primary human fetal neurons were fixed in 4% paraformalde-hyde (PFA), blocked in buffer containing 0.4% Triton X-100,and incubated overnight at 4�C with primary antibody.Immunocytochemical tests were examined using a Nikon(Tokyo, Japan) Microphot-FXA microscope. For the quantita-tion of immunoreactive cells, 20 randomly selected fields wereanalyzed.

Optic nerve crush injury

The optic nerve was subjected to crush injury as described[38]. Briefly, rats were deeply anesthetized by intramuscularinjection of XYL-M 2% (xylazine, 10 mg/kg; Arendonk,Belgium) and Ketaset (ketamine, 50 mg/kg; Fort DodgeLaboratories, Fort Dodge, IA). Using a binocular operatingmicroscope, temporal canthotomy was performed in the righteye, and the conjunctiva was incised lateral to the cornea. Carewas taken to preserve retinal blood supply. After separation ofthe retractor bulbi muscles, the optic nerve was exposedintraorbitally by blunt dissection. Using calibrated cross-actionforceps, the optic nerve was subjected to a ten-second crushinjury 1–2 mm from the eye. The uninjured contralateral nervewas left undisturbed.

Measurement of secondary degeneration by retrogradelabeling of retinal ganglion cells

Two weeks after crush injury fluorescent lipophilic dye, 4-(4-(didecylamino)styryl)-N-methylpyridinium iodide (4-Di-10-Asp) (Molecular Probes Europe BV, The Netherlands) wasapplied distal to the lesion site. Five days after dye applicationthe retinas were detached from the eye, prepared as a flattenedwhole-mount in a 4% paraformaldehyde solution, andexamined for labeled (retinal ganglion cells) RGC using

fluorescence microscopy. RGC count was performed on 16fields for each retina located equidistant from the optic nervehead. Counts were performed under 40x magnification by aninvestigator who was masked to the treatment groups. At least10–12 fields were counted from each retina located at an equaldistance from the optic head.

Acknowledgements: We thank Bonnie Bloch andShirley Smith for editing the manuscript and R. Taylorfor graphical assistance. The work was supported inpart by Nebraska Tobacco Settlement BiomedicalResearch Development Fund and in part by a PilotAward grant from National Multiple Sclerosis Society(PP1221) to J.K.

Conflict of interest: All authors disclaim financialconflict of interests.

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T cell independent mechanism for copolymer-1-induced neuroprotection

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